Ficoll and dextran enhance adhesion of Sendai virus to liposomes containing receptor (ganglioside GD1a)

Previous work has shown that high-speed centrifugation (300,000 g) of Sendai virus and liposomes in 40% (w/v) sucrose layered under a discontinuous sucrose gradient removes Sendai virus bound to liposomes containing the ganglioside GD1a, a Sendai virus receptor. Centrifugation also removes virus bound to liposomes containing other negatively charged lipids. This work shows that centrifugation of virus through a discontinuous ficoll gradient does not remove virus bound to liposomes containing GD1a but does remove virus from liposomes containing various other negatively charged lipids including the ganglioside GM1, which is not a Sendai virus receptor. The amount of virus that adheres to liposomes increases with increasing content of GD1a in the liposomes. The adhesion of virus to receptor-containing liposomes during centrifugation through a ficoll gradient results from the presence of ficoll and increases with increasing ficoll concentration. Virus also adheres to receptor-containing liposomes during centrifugation in the presence of dextran. These data indicate that caution should be used in interpreting associations demonstrated by centrifugation through dextran and ficoll gradients. They also indicate that binding of virus by ganglioside receptors can be modulated by carbohydrate polymers, which are thought not to have any specific interaction with either viruses or gangliosides.     

Receptor ganglioside content of three hosts for Sendai virus. MDBK, HeLa, and MDCK cells

Specific gangliosides GD1a, GT1b and GQ1b isolated from brain have been shown to function as receptors for Sendai virus by conferring susceptibility to infection when they are incorporated into receptor-deficient cells (Markwell, M.A.K., Svennerholm, L. and Paulson, J.C. (1981) Proc. Natl. Acad. Sci. USA 78, 5406-5410). The endogenous gangliosides of three commonly used hosts for Sendai virus: MDBK, HeLa, and MDCK cells were analyzed to determine the amount and type of receptor gangliosides present. In all three cell lines, GM3 was the major ganglioside component. The presence of GM1, GD1a and the more complex homologs of the gangliotetraose series was also established. In cell lines derived from normal tissue, MDBK and MDCK cells, gangliosides contributed 47-65% of the total sialic acid. In HeLa cells, gangliosides contributed substantially less (17% of the total sialic acid). The ganglioside content of each cell line was shown not to be immutable but instead to depend on the state of differentiation, passage number, and surface the cells were grown on. Thus, the ganglioside concentration of undifferentiated MDCK cells was found to be substantially greater than that of MDBK or HeLa cells, but decreased as the MDCK cells underwent differentiation. Changes in culture conditions that were shown to decrease the receptor ganglioside content of the cells resulted in a corresponding decrease in susceptibility to infection. The endogenous oligosialogangliosides present in susceptible host cells were shown to function as receptors for Sendai virus.     

Characteristics of Sendai virus receptors in a model membrane

The adsorption of Sendai virus to liposomes of different compositions was studied. Liposomes prepared with only phosphatidylcholine and cholesterol, and liposomes prepared with phosphatidylcholine and cholesterol plus phosphatidic acid or phosphatidyl serine did not adsorb virus. Phosphatidyleholine-cholesterol liposomes containing also stearyl amine or ganglioside did, however, adsorb virus. The ability of the adsorbing liposomes to compete with erythrocytes for virus was measured by hemagglutination inhibition. Liposomes containing ganglioside, but not those containing stearyl amine, inhibited hemagglutination. When the molar ratio of ganglioside N-acetyl neuraminic acid to phosphatidylcholine was less than 0.02, ganglioside liposomes did not inhibit hemagglutination. As the ratio increased from 0.02 to 0.05, the liposomes caused increasing amounts of hemagglutination inhibition, but with further increases in the ratio the hemagglutination inhibition remained constant. It is concluded that gangliosides can serve as Sendai receptors and that a multiplicity of receptors is needed for virus binding.     

Action of ortho- and paramyxovirus neuraminidase on gangliosides. Hydrolysis of ganglioside GM1 by Sendai virus neuraminidase

The action of neuraminidase of influenza A virus, Sendai virus and Newcastle disease virus particles on bovine brain ganglioside GM1 and the properties of Sendai virus neuraminidase for GM1 were studied. With Sendai virus, GM1 was hydrolyzed to asialo-GM1 (GA1) and N-acetylneuraminic acid even in the absence of surfactant or other additives, while the hydrolysis of GM1 by Newcastle disease virus or influenza A virus was very low or undetectable under the same conditions. The formation of GA1 by Sendai virus neuraminidase was confirmed by thin-layer chromatography and immunodiffusion test using anti-GA1 antiserum. The apparent Km of Sendai virus neuraminidase for GM1 hydrolysis was found to be 2.67 x 10(-4) M and the optimum pH was 5.6. GM3, GM2 and oligosaccharide of GM1 were hydrolyzed more effectively than GM1 in the absence of surfactant (GM3 greater than GM2 greater than oligosaccharide of GM1 greater than GM1). The hydrolysis of GM1 by the Sendai virus enzyme was stimulated by the addition of sodium cholate or sodium taurocholate, but was inhibited by divalent cations (10 mM), Ca2+, Mg2+, ZN2+, Fe2+ and CU2+. In the absence of the surfactant, Sendai virus neuraminidase hydrolyzed GM1 more efficiently than Arthobacter ureafaciens neuraminidase which has been reported recently as being an adequate enzyme to hydrolyze ganglioside GM1 as a substrate.     

Sendai virus induced leakage of liposomes containing gangliosides

Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic and phase characterization of phosphatidylethanolamine and ganglioside GD1a mixtures

By employing diphenylhexatriene steady-state fluorescence anisotropy, pyrenedecanoic acid excimer formation, and high sensitivity scanning calorimetry we have demonstrated that the liposomes containing phosphatidylethanolamine (PE) and various mole fractions of ganglioside GD1a had a gel-liquid crystalline phase transition between 15 and 25 degrees C. Calorimetric measurements indicated that these phase transitions were broad and centered between 17 and 21 degrees C. The enthalpy change of the transition was linearly dependent on the ganglioside concentration up to 10.0 mol% and plateaued between 11.4-16.2 mol%. The high enthalpy change (37 kcal/mol of GD1a added into the PE bilayer) indicates the existence of PE-GD1a complex structure in the liposomal membrane. It is proposed that semi-fluid domains containing six PE and one ganglioside molecule are present in the PE-GD1a membranes at temperatures above gel-liquid crystalline phase transition. The Sendai virus induced leakage of PE-GD1a liposomes has been investigated by using an entrapped, self-quenching fluorescent dye, calcein. The leakage rate was dependent on the mole fraction of ganglioside GD1a and was maximal at 6.3 mol%. Arrhenius plots of the leakage rates showed breaks in the 20-25 degrees C temperature range, which correspond to the gel-liquid crystalline phase transition of the target liposomes. These data suggest that the rate of Sendai virus-induced leakage can be regulated via fluidity modulation by changing the PE to GD1a ratio at constant temperatures.     

Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration

The conditions that optimize Sendai virus membrane fusion with liposomes have been studied. No fusion occurs in the absence of ganglioside receptors. Maximum fusion occurs when the molar ratio of ganglioside GD1a to phospholipid is 0.02 or greater. The amount of fusion at 37 degrees C increases with time up to at least 6.5 h. The rate of fusion increases from the lowest temperature tested, 10 degrees C, to 40 degrees C. Above 43 degrees C the amount of fusion decreases because of thermal inactivation of the viral proteins. There is a broad pH maximum between pH 7.5 and pH 9.0. At both ends of the pH range the amount of fusion increases and exceeds that found in the physiologic pH range. Neither ethylenediaminetetraacetic acid nor Ca2+ changes the amount of membrane fusion. The optimal conditions for membrane fusion of Sendai virus membranes with liposomes are the same as the optimal conditions for fusion with host cells and with red blood cells. Since the liposomes contain no proteins, the optimal conditions for Sendai virus membrane fusion must be determined by the viral proteins and be mostly independent of the nature or presence of the host proteins.     

A protease activation mutant, MVCES1, as a safe and potent live vaccine derived from currently prevailing Sendai virus.

Sendai virus fresh isolates were shown to be antigenically different from the prototype Fushimi strain that had long been passaged in embryonated chicken eggs. Phylogenetic analysis of the hemagglutinin-neuraminidase genes also revealed the difference between these two virus groups. Both trypsin-resistant and elastase-sensitive mutations were additionally introduced to an LLC-MK2-cell-adapted and attenuated mutant derived from one of the fresh isolates. This protease activation mutant (MVCES1) showed the same antigenicity as the fresh isolates, and as a result of a single cycle of growth in lungs, it could confer better protection on mice against challenge infection with the currently prevailing Sendai virus than TR-5, which is a trypsin-resistant mutant derived from the Fushimi strain. The eligibility of MVCES1 as an attenuated live vaccine of Sendai virus is discussed.     

Thermodynamic and phase characterizations of phosphatidylethanolamine and ganglioside GD1a mixtures

By employing diphenylhexatriene steady-state fluorescence anisotropy, pyrenedecanoic acid excimer formation, and high sensitivity scanning calorimetry we have demonstrated that the liposomes containing phosphatidylethanolamine (PE) and various mole fractions of ganglioside G_D1a had a gel-liquid crystalline phase transition between 15 and 25° C. Calorimetric measurements indicated that these phase transitions were broad and centered between 17 and 21° C. The enthalpy change of the transition was linearly dependent on the ganglioside concentration up to 10.0 mol% and plateaued between 11.4–16.2 mol%. The high enthalpy change (37 kcal/mol of G_D1a added into the PE bilayer) indicates the existence of PE-G_D1a complex structure in the liposomal membrane. It is proposed that semi-fluid domains containing six PE and one ganglioside molecule are present in the PE-G_D1a membranes at temperatures above gel-liquid crystalline phase transition. The Sendai virus induced leakage of PE-G_D1a liposomes has been investigated by using an entrapped, self-quenching fluorescent dye, calcein. The leakage rate was dependent on the mole fraction of ganglioside G_D1a and was maximal at 6.3 mol%. Arrhenius plots of the leakage rates showed breaks in the 20–25° C temperature range, which correspond to the gel-liquid crystalline phase transition of the target liposomes. These data suggest that the rate of Sendai virus-induced leakage can be regulated via fluidity modulation by changing the PE to G_D1a ratio at constant temperatures.     

Stimulation and modulation of adenylate cyclase by Sendai virus.

Stimulation of adenylate cyclase activity occurs in membranes prepared from toad erythrocytes preincubated briefly (at 37° or 4°) with ultraviolet light-inactivated Sendai virus. Stimulation occurs with as few as five virions per cell, and it is blocked by pretreating the virus with the membrane glycolipid, ganglioside GM₁. Virus treatment also alters modulation of adenylate cyclase by hormones, nucleotides and sodium fluoride. Interactions of viral envelope antigens with plasma membrane components may thus elicit functional changes possibly important in the pathogenesis of viral infections.     

Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se.     

The relationship between prostaglandins and virus replication: Endogenous prostaglandin synthesis during infection and the effect of exogenous PGA on virus production in different cell lines and in persistently infected cells

African Green Monkey Kidney cells were shown to normally synthesize immunoreactive PGE₁. Infection of these cells with Sendai virus did not alter rates of PGE₁ synthesis, while it stimulated interferon production. PGAs, that we have previously shown to be potent inhibitors of Sendai virus replication in this system, at the same dose (4 μg/ml), also strongly inhibited the replication of this virus in HEp-2 cells and in VERO cells, a monkey kidney cell line that does not produce interferon. PGA₁ was found to be effective in several cell and virus models, suggesting a broad spectrum of antiviral actions. Finally, we confirmed the observation that PGA₁-treatment prevents the establishment of a “carrier state” by Sendai virus, and PGA₁-cured cells did not show any sign of persistent infection for periods as long as 110 days after Sendai function. Attempts to cure already established persistently infected cells were only partially successful.     

Effect of N-acyl-phosphatidylethanolamine on the Membrane Fusion Between Sendai Virus and Liposome

We have compared the properties of two N-acyl derivatives of dilauryl phosphatidylethanolamine on lipid polymorphism, vesicle leakage and Sendai virus fusion. The derivatives contained either an N-lauroyl group (NLPE) or an N-acetyl group (NAcPE). Only the NAcPE markedly affected the bilayer to hexagonal transition temperature of dielaidoyl phosphatidylethanolamine, shifting it to higher values. In contrast the NLPE slightly lowered this phase transition temperature. The two lipids also have opposite effects on leakage from small unilamellar vesicles of egg phosphatidylcholine. The NLPE inhibits leakage, while the NAcPE promotes it. This vesicle stabilizing effect of NLPE against leakage is not manifested in alterations of rates or extents of Sendai virus fusion to liposomes of egg phosphatidylethanolamine plus 2% ganglioside G_D1a. The NLPE has no effect, while the NAcPE reduces the observed fusion, at least in part as a consequence of a reduction in the final extent of fusion. These results demonstrate that the bilayer stabilizing effects of NLPE do not result in a lower rate of viral fusion. Furthermore, these bilayer stabilizing effects against leakage are not solely a function of the lipid headgroup but also require a structure with three long acyl chains. The reduced leakage is not related to a loss in monolayer curvature strain.     

Fusion-mediated injection of SV40-DNA : Introduction of SV40-DNA into tissue culture cells by the use of DNA-loaded reconstituted Sendai virus envelopes

Sendai virus envelopes can be solubilized by non-ionic detergents such as Triton X-100. Removal of the detergent from a supernatant containing the solubilized viral envelope glycoproteins results in the formation of reconstituted fusogenic viral envelopes. When SV40-DNA is added to the reconstitution system, it is trapped within the viral envelope. Incubation of SV40-DNA-loaded Sendai virus envelopes with permissive cells (CV1 and TC7 cells) resulted in fusion-mediated injection of the trapped DNA, as was demonstrated by the ability of the injected cells to synthesize SV40-T-antigen. Quantitative estimation revealed that up to 20% of the injected cells were able to synthesize T-antigen. Loaded viral envelopes were able to inject SV40-DNA and to promote synthesis of T-antigen also in cells which are resistant to infection by intact SV40 viruses, such as F1' 1-4 cells. In addition, it is shown that reconstituted envelopes of Sendai virus are able to transfer membrane fragments from SV40 receptor-positive into SV40 receptor-negative cells, such as F1' 1-4 cells. After implantation of SV40 receptors, the F1' 1-4 cells became susceptible to infection by intact SV40 viruses.     

Loss of reactivity of a myeloma tumor with H-2 compatible thymus-derived lymphocytes.

It was previously shown that MOPC-315-EL, a subline of the BALB/c myeloma tumor MOPC-315, reversibly alters its reactivity with T cells that recognize H-2^d, 2,4,6-trinitrophenyl, and minor histocompatibility antigens. This report demonstrates (a) that CTLs directed against vesicular stomatitis virus and Sendai virus are unable to recognize viral antigens associated with the unreactive tumor cell, and (b) that incorporation of Sendai virus antigens into the same membrane as the H-2 gene products is required for effective recognition by cytotoxic T lymphocytes.     

Detection of antibodies to Sendai virus in mouse sera by enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) for diagnosis of Sendai virus infection in mice was evaluated. A large-scale survey of infected mice showed that ELISA is approximately 100 times more sensitive than the hemagglutination-inhibition or complement-fixation test. Although a few SPF mice showed false-positive reactions at a serum dilution of 1:40, further dilution to 1:80 eliminated the non-specific reaction. It was shown that ELISA is a highly satisfactory method for examination of Sendai virus infection in mice.     

Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus

A sensitive assay system for receptor activity of gangliosides to paramyxovirus was developed. This system involves incorporation of gangliosides into neuraminidase-treated chicken erythrocytes (asialoerythrocytes) followed by estimation of virus-mediated agglutination and hemolysis. The asialoerythrocytes coated with I-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer) were effectively agglutinated by hemagglutinating virus of Japan (HVJ, Sendai virus). The hemolysis of the asialoerythrocytes mediated by HVJ was restored to the highest level by labeling the cells with gangliosides possessing lacto-series oligosaccharide chains, i.e., I-active ganglioside, N-acetylneuraminosylparagloboside (SiaPG(NeuAc)), and i-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer). The specific receptor activity of ganglioside GD1a possessing a gangliotetraose chain was lower than those of the gangliosides described above. Gangliosides GM3, GD3, GM1a, GD1b, SiaPG(NeuGc) showed little effect on the restoration of HVJ-mediated hemolysis. On infection with Newcastle disease virus (NDV), the highest specific restoration of lysis was found in chicken asialoerythrocytes coated with SiaPG(NeuAc or NeuGc) and GM3(NeuAc or NeuGc), whereas those coated with I-active ganglioside, GD3, GM1a, and GD1b showed very low NDV-mediated hemolysis. The above results indicate that the determinants of receptor for HVJ contain sialylated branched and/or linear lacto-series oligosaccharides carried by I,i-active gangliosides and SiaPG(NeuAc) and sialosylgangliotetraose chain carried by GD1a. The determinants for NDV are carried by SiaPG(NeuAc or NeuGc) containing linear lacto-series oligosaccharide and GM3(NeuAc or NeuGc). The absence of detectable binding of free oligosaccharides obtained from I-active ganglioside and sialoglycoprotein GP-2 isolated from bovine erythrocyte membranes as HVJ receptor (Suzuki, Y., et al. J. Biochem. (1983) 93, 1621-1633; (1984) 95, 1193-1200) indicates that HVJ recognizes the sialooligosaccharides oriented out of the lipid bilayer in the cell membranes where the hydrophobic ceramide or peptide backbone of the receptor is integrated.     

Analysis of a Viral Agent Isolated from Multiple Sclerosis Brain Tissue: Characterization as a Parainfluenzavirus Type 1

A virus originally isolated from cell cultures obtained by lysolecithin-induced fusion of human multiple sclerosis brain cells with CV-1 cells has been analyzed for its antigenic, RNA, and polypeptide compositions, and for selective biological properties. Our findings establish that this isolate, designated 6/94 virus, contains a 50S RNA genome and is, as yet, indistinguishable from Sendai virus in its antigenic and total polypeptide compositions. Despite these similarities, the 6/94 and Sendai viruses differ in certain phenotypic properties. 6/94 virus is markedly less cytocidal for chick fibroblasts, especially at 37 C and, after beta-propiolactone inactivation, it possesses a greater capacity for cell fusion and a lower toxicity than does comparably treated Sendai virus. In addition, 6/94 virus shows greater hemolytic activity.     

N-glycolyl GM1 ganglioside as a receptor for simian virus 40

Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.